IL47594A - Process for obtaining protein-dyestuff conjugates - Google Patents

Description

-DYESTUPF PROCESS FOR OBTAINING PROTEIN^"CONJUGATES (HOE 7 /B 011) Protein-dyestuff conjugates, especially immune sera containing antibodies and conjugated with fluorescent dyes, are^ particularly important in the so-called immunofluorescence technique, as will be shown in the following without limiting the invention to these examples.

The immunofluorescence technique, reported by A.H. Coons in 19^2, permits a combined investigation of serologic-immunologic specificity and of the morphological characteristics of the material to be analyzed. This method is based on the so-called antigen-antibody reaction, in which one of the two immunologic components - generally, the antibody - is conjugated with a fluorescent dyestuff. The anti^gen-antibody reaction Compound is made visible in the microscopic preparation by provoking the visible fluorescence by means of rays in the ultraviolet range.

The immunofluorescence technique has acquired great significance in the diagnosis for identifying linked and free antigens and antibodies.

According to the slate of the art, fractions with a high concentration of antibodies are reacted as so-called immune sera with fluorescent dyes, preferably with fluorescein iso-thiocyanate. The reaction product is then predominantly purified by gel filtration and by ion exchange chromatography on diethyl-aminoethyl (DEAE) cellulose, whereby the fluorescein-conjugated immune globulins are isolated.

Those known methods have the drawback that the separation of the dyestuff, which is not linked by a covalent bond, from the fluorescein-conjugated protein composition by means of gel filtration or dialysis is incomplete; in particular, a dyestuff which is linked to protein by adsorption or by a weak covalent bond that can relatively easily be split up by hydrolysis, i not eliminated. Moreover, a dyestuff which reaches a high molecular weightp owing to autopolymerization or autocon-densation, is also eliminated to an unsatisfactory extent only,; Compositions containing such free dyes lead to unspecific fluorescence colorations in the microscopic preparation and thus to false results.

The widely used prior art process for obtaining conjugated protein fractions containing antibodies by means of ion exchange chromatography on DEAE cellulose generally provides only the antibody fraction belonging to the IgG class. Under the conditions applied for elution and separation, the antibodies of the IgM and IgA classes are separated only unsatis-factorily or are not separated at all, so that the preparation obtained partly shows a substantial loss of the desired specific antibodies. When modifying the elution conditions suitable for the recovery of fluorescein-conjugated IgM and IgA antibodies, so-called "overlabeled" 2T~S^otu^^ns as weH as baso-philic proteins are also eluted. Owing to their strongly acid nature, these "overlabeled" protein fractions lead, in the immunologic reaction, to an unspecific linkage to a number of basic proteins in the material to be examined and thus again to a misinterpretation of the fluorescence results.

The process of this invention avoids those drawbacks. The electrophoretic purification of dyestuff-conjugated protein preparations, preferably fluorescein-conjugated protein preparations, carried out preferably in an inert carrier material after the conjugation reaction, offers the following advantages; The process permits an optimum recovery of "accurate" zon s containing antibodies. Undesired protein fractions that a^, for example, free of antibodies can easily be eliminated together with the separation of the desired fraction.

The yield of the dyestuff-conjugated protein preparations containing antibodies is optimum. In an exemplified case of a fluorescein-labeled ^-globulin fraction, 70 to 90 % of the amount used are recovered.

The conjugation reaction of proteins with acidic organic molecules reduces the equipotential points in all the proteins reacted with the organic molecules, so that under the electrophoresis conditions, the reaction products migrate toward the anode at a higher speed than non-conjugated proteins.

There is no difficulty in eliminating proteins which have become too acidic owing to an intensive reaction with the molecule used for conjugation, since these migrate toward the anode at a higher speed. This finding is especially significant with the proteins that are conjugated with fluorescein isothiocyanate as the acidic fluorescent dyestuff (quinoid system with a free carboxylic group).

The process of the invention ensures the optimum separation of the acidic organic molecule, which is not linked (or linked by adsorption) and which, if present in the fluores-cein-conjugated compositions, leads to undesired, unspecific fluorescence colorations. The reason for this easy elimination of this dyestuff portion is that the acidic molecule itself has a strong tendency to migrate toward the anode.

The electric potential established under the conditions of the preparative zone electrophoresis causes the molecules which are relatively loosely bound to protein to be split off and enhances the quality of the protein conjugates recovered^ by electrophoresis. In the case of proteins conjugated with fluorescent dyes, the stability of such preparations, when stored in an aqueous solution, is increased in comparison with those obtained according to the state of the art. 4. When antibodies are used as proteins for the conjugation, the process of the invention allows all the dyestuff-con- jugated antibody classes to be obtained, for example IgA, IgG, IgM. This is of importance especially for the use of antibodies obtained from sera, for example of man, goat, sheep and horse, in whlch-besides the antibodies of the IgG class-also antibodies of other immunoglobulin classes account for a relatively high percentage of the tdal antibody amount. This factor is also of a substantial economic interest since antibody-containing sera are available to only a limited extent.

Used for the purification of antibodies conjugated with fluorescent dyes, the process of the invention brings about an increase in the relationship between specific antibodies (Ab) and total protein (P), the so-called Ab/P quotient, which again intensifies the desired specific fluorescence effect. 5. The process, which can be operated without using expensive chromatographicai adjuvents, results in a substantial cost reduction for the manufacture of pure protein conjugates. The inert carrier material used may be employed several times.

The preferable electrophoretic method used is the carrier electrophoresis, in particular the electrophoresis using the inert carrier material in a horizontal container. When, for example, polyvinyl chloride granules are used as the carrier material, it is possible after the separation of the fluores-celn-conjugated protein fractions containing the antibodies to cut out the desired preparation in accordance with the visible bands and then elute it from the carrier by means of simple solvents.

When the conjugate is uncolored, the positions of protein, reaction component and protein conjugate can be established indirectly, for example by means of a paper dabbing which is then colored in known manner.

In addition to polyvinyl chloride, for example polyacryl-amide, cellulose, starch, glass beads, sand and the like are frequently used as electrophoretic carrier materials.

Electrophoresis equipment used according to the invention also include vertical arrangements using carrier material or not. Very good purification effects are also obtained using continuously operating equipment.

The process of this invention separates antibody preparations of any origin, which have been linked by covalent bond to dyes of acidic nature, especially to fluorescein isothiocyanate.

It is, however, also possible to react non fluorescent dyes with proteins and to separate the proteins colored by the conjugation reaction according to the process of the invention, provided the dyes used show different electrophoretic migration properties.

Instead of antibodies, other proteins of animal, vegetable and microDial origin may also be used, if they exhibit as con-Jugates an electrophoretical property that differs from that of the starting protein and of the organic molecule used for the conjugation. Organic molecules preferably used for the conjugation are compounds having their equlpotential points within the acid pH-range.

The essential feature of this invention resides in applying preparative electrophoretic methods for the manufacture of pure protein conjugates, especially of gamma-globulin fractions reacted with fluorescent dyes. The reaction of ^f-globulin fractions with conventional fluorescent dyes is not expected to yield an electrophoretically homogeneous product, since the structures of the immunoglobulin classes used for the reaction are known to be different and rather have a different number of amino groups available for the reaction. Although electrophoretical methods are known to provide relatively uniformly migrating zones of unreacted ^-globulins, the uniform migration of immunoglobulins reacted with dyes and belonging to different classes has to be regarded as a surprise. Moreover, the dyes linked to protein molecules by adsorption could not be expected to be separated from the protein in an electric field. Finally, owing to the known intense linkage of fluorescent dyes to ion exchange material as used in the state of the art, it could not be foreseen that the free dyestuff used in an electrophoretic method, especially together with polyvinyl chloride as a carrier material, migrates over a particularly long distance as compared to that of the protein conjugates.

The known process of electrophoresis yields a product of substantially higher purity than chromatographical methods do.

The product obtained exhibits, as a surprising technical effect, a higher specificity when used as an immunological reagent th the product obtained according to the state of the art.

The following Examples illustrate the invention.

E X A M P L E 1: A 3 % antibody-containing serum protein solution (goat) was reacted in known manner with fluorescein; isothiocyanate in an alkaline medium. In a horizontal electrophoresis equipment (size: 65.0 cm in length, 76.0 cm in width, and 1.2 cm in height), which contained 3.6 1 of PVC powder (Geon X 427, trade mark of Messrs. Serva, Heidelberg, W.Germany), the conjugated antiserum was separated at pH 8.1, at a field potential of 6 V per cm, and at an amperage of 0.6 A, over a period of 15 hours. The ^-globulin fraction containing the antibodies, which generally remains unmoved at the application point or even migrates toward the cathode unless conjugated to fluorescein isothiocyanate, migrated clearly toward the anode. The light yellow to greenish coloration of the ^-globulin fraction containing the antibodies stood clearly out against the rest of protein fractions and free dyestuff portions with their dark yellow to reddish brown color. The desired ^-globulin fraction containing the antibodies and having the optimum F/P quotient -which can also be determined bandwise immediately after separation - was cut out as a PVC zone and washed clear of the carrier material by means of a 0.9 % sodium chloride solution. The eluate was concentrated to about 1 g % of protein by ultrafiltration.

This last operation was followed by the final standardization and quality control of the product.

From 3.6 g of a fluorescein-conjugated protein solution containing antibodies (120 ml of antiserum), 0.5 g of a fluor¾s-cein-conjugated fraction containing antibodies was obtained.

When serum proteins of rabbits containing antibodies were used in an amount of 8 g of a fluorescein-conjugated antiserum containing antibodies (120 ml), 1.0 g of a fluorescein-con-jugated ^-globulin fraction containing antibodies was obtained.

Analogous yields were obtained by recovering antibodies from human sera. Considering that the proportion of the ^-globulin fraction in an antiserum accounts for about 15 to 18 %, these yields, calculated on this ^-globulin fraction, can be regarded as optimum.

E X A M P L E 2; A 3 % human transferrin solution was reacted in known manner with fluorescein isothiocyanate in an alkaline medium.

In a horizontal electrophoresis equipment (size: 65.0 cm long, 76.0 cm wide, and 1.2 cm high) containing 3.6 1 of a PVC powder (Geon X 427, trade mark of Messrs. Serva, Heidelberg, W.Germany), a conjugated protein was separated at pH 8.1, at; a field potential of 6 V per cm and at an amperage of 0.6 A, over a period of 15 hours. The transferrin conjugate clearly migrated toward the anode. The yellow-brown coloration of the trans-ferrin-containing zone stood clearly out against the different colors of the free dyestuff portions which had migrated still further toward the anode. The transferrin conjugate having the optimum F/P quotient - which can be determined bandwise immediately after separation - was cut out as a PVC zone and washed clear of the carrier material by means of a 0.9 % sodium chloride solution. The eluate was concentrated to about 1 g of protein by ultra-filtration.

This operation was followed by the final standardization and quality control of the product.

From 3.6 g of human transferrin, about 3.0 g of the corresponding fluoresceln-conjugated protein were obtained.

Claims (5)

1. HOE 7 /B 011 47594/2 We claim: -!yestuff A process for the purification of a protein/ conjugate which differs from the organic compound used for the conjugation in its electrophoretical properties, which process comprises subjecting the crude

2. A process as claimed in claim 1 , wherein an antibody-containing gamma-globulin fraction that has been conjugated with a dye is purified.

3. A process as claimed in claim 1 , wherein an antibody-con- taining gamma-globulin fraction that has been conjugated with a fluorescent dye is purified. A process as claimed in claim 1 , wherein an electrophoretic method using an inert carrier material is applied. dyestuff A protein /conjugate; that has been purified according to the process claimed in claim . r

4. 6. A method of immunological diagnosis, wherein one of the reagents is a protein-dyestuff conjugate as claimed in claim

5. Attorneys for Appl C u at